Bats experience age-related hearing loss (presbycusis)

Bats exhibited age-related hearing loss. Bats are exposed to immense conspecific noise, but the frequency-dependent hearing loss was not correlated to the noise.

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Thank you for this interesting contribution to Life Science Alliance. We are looking forward to receiving your revised manuscript. --High-resolution figure, supplementary figure and video files uploaded as individual files: See our detailed guidelines for preparing your production-ready images, https://www.life-science-alliance.org/authors --Summary blurb (enter in submission system): A short text summarizing in a single sentence the study (max. 200 characters including spaces). This text is used in conjunction with the titles of papers, hence should be informative and complementary to the title and running title. It should describe the context and significance of the findings for a general readership; it should be written in the present tense and refer to the work in the third person. Author names should not be mentioned.
--By submitting a revision, you attest that you are aware of our payment policies found here: https://www.life-sciencealliance.org/copyright-license-fee B. MANUSCRIPT ORGANIZATION AND FORMATTING: Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** ---------------------------------------------------------------------------Reviewer #1 (Comments to the Authors (Required)): In this work, Tarnovsky et al. want to determine whether Egyptian fruit bats, echolocating animals naturally exposed to frequent high-intensity sound, are subjected or not to age-related hearing loss like mammals. They assessed the hearing of 47 Egyptian fruit bats population by ABR and CM while estimating their age by methylation profiling. Based on their results, they suggest that: -The occurrence of 10 dB age-hearing loss in bats between groups of 2.8 years old average age and 12 years old animals.
-An increased rate of hearing loss with age with higher frequencies -The defect is both peripheral, with a reduction of ABR and CM thresholds, and central, with an increased latency between central auditory relays.
While this study is solid and provides some important results, some points need to be addressed, corrected, or improved: Major points: -The main results about the ABR thresholds at different ages and different frequencies, and the slope analysis should be improved: It is difficult to determine the quality of the slope fit. Please provide statistical analysis to evaluate this fit. Also, I suggest showing, potentially in a supplemental figure, the average threshold for each frequency along with the fit. I understand that the older animals were rarer. Still, one needs to be able to determine how the results from the 2 oldest animals fit compared to the one before because they will drastically influence the fit. While it could occur faster for higher frequencies, the data show a similar magnitude of hearing loss across frequencies, which differs from mouse and human ARHL.
-The analysis of gender biases seems not possible for single or partially grouped ages because of sampling differences: o 2-5 years old: 15 males vs 7 females o 6-8 years old: 3 males vs 14 females o 10-14 years old: 4 males vs 6 females -It is a missed opportunity not to have analyzed the entire frequency spectrum of the environmental noise exposure of the bats to test if there is a correlation with the rate of age-related HL. The noise intensity contribution per frequency should be plotted as distributions for a better view. Also, what is the hearing range of these animals? Why has a very low frequency not been included? -How the CM have been recorded is unclear to me. An electrode is usually placed next to the tympanic membrane or into the scala tympani. Please provide a detailed description of the procedure.
-The cochlear histology should be improved: Spiral gg neuron density and the presence of hair cells in the epithelium along the tonotopical axis should be imaged (from histology) and quantified. All results could originate from the degenerescence of hair cells and spiral ganglion neurons.
Minor: -Different fonts in the sentence : "In addition, we recorded cochlear microphonics (CM) noninvasively, using the same subdermal electrode positioning used for the ABR recordings. The CM response represents activity from the outer hair cells (OHC), which amplify the sound-induced motions in the inner ear, while the inner hair cells (IHC) translate these motions into the chemical signals that excite the auditory nerve (AN) (Liberman, 2015). " -In significance statement, space missing between <10 gr -(SPL) Re 20 Pa : what "Re" means? -what is a GLME? -Check space between value and unit (ex 8kHz shoukd be 8 kHz) Reviewer #2 (Comments to the Authors (Required)): Referee comments on LSA-2022-01847-T Bats suffer from age-related hearing loss (presbycusis) Tarnovsky et al. This is an ambitious study using multiple methods to assess the occurrence and magnitude of age-related hearing loss in Egyptian fruit-bats, Rousettus. The use of new DNA methods to assess age in bats, and the use of minimally-invasive neurophysiological recording techniques are useful methodological advances. So is the use of cochlear microphonic recordings.
First, I suggest changing the title and any use of the term, "suffer," to describe presbycusis in Rousettus. I suggest using "Bats experience.." instead of "Bats suffer from..." throughout the manuscript. "Suffer" implies punishing effects or debilitating consequences that are not observed, and that brings to mind negative connotations to readers that are not good to evoke. The measured sge-related hearing losses amount to only about 1 dB per year. Second, I suggest changing "noninvasive" to "minimally-invasive" for the ABR and CM recordings in unanesthetized bats. The Simmons et al 2022 citation makes this distinction desirable. The absence of page numbers or line numbers in the manuscript make it difficult to specify further suggestions, but the pdf reader does give pages. Page 4, second paragraph: Linnenschmidt & Wiegrebe, 2019 is not a good general reference to echolocation for readers, so I suggest the two recent Springer books Biosonar and Bat Bioacoustics. Page 4, Last paragraph: to the citations of Kick & Simmons, 1984;Liu et al., 2021, add Simmons et al 2016 This reference seems to be the earliest to directly address noise damage effects on hearing and then actually test it behaviorally. Page 6, first paragraph: cochlear microphonics (CM) seems to be a in different text font than the rest of the paragraph. Pages 27-28: CM recordings were cleverly done using short clicks and a 30-cm speaker-to-ear tube, plus phase reversals to ensure the recordings were really physiological and not speaker radiation artifacts, which are the most difficult problem for CM recordings.
1st Authors' Response to Reviewers February 4, 2023 We thank the reviewers for these comments.

We added page and line numbers.
Changes in the manuscript and the supplementary file are in red letters.

Reviewer #1
Major points: -The main results about the ABR thresholds at different ages and different frequencies, and the slope analysis should be improved: It is difficult to determine the quality of the slope fit.
Please provide statistical analysis to evaluate this fit.

We added the P values of the slopes in the figure's legend (page 8).
Also, I suggest showing, potentially in a supplemental figure, the average threshold for each frequency along with the fit.
We added the thresholds in figure 1D -as blue dots.
I understand that the older animals were rarer. Still, one needs to be able to determine how the results from the 2 oldest animals fit compared to the one before because they will drastically influence the fit. While it could occur faster for higher frequencies, the data show a similar magnitude of hearing loss across frequencies, which differs from mouse and human ARHL.
We are not sure that we understand this comment. First, we note that we did see a higher decrease in hearing at higher frequencies and second, we show all of the thresholds for all bats (of all ages) in figure 1D. We tried to better clarify this in the legend. We apologize if we are missing the reviewer's suggestion.
-The analysis of gender biases seems not possible for single or partially grouped ages because of sampling differences: We ran a generalized linear model (GLM) with all ages together and found no effect of sex. To our best statistical undertanding, this is legitimate as the model takes all individuals into account together. In the revised mns we also examined a model that includes sex-age interaction and found no significant difference in the model' BIC.
-It is a missed opportunity not to have analyzed the entire frequency spectrum of the environmental noise exposure of the bats to test if there is a correlation with the rate of agerelated HL. The noise intensity contribution per frequency should be plotted as distributions for a better view. Also, what is the hearing range of these animals? Why has a very low frequency not been included?
Following the reviewe's comment, we estimated noise exposure at all relevant frequencies and examined the correlation with hearing loss. We found an inverse correlation between the rate of age-related hearing loss and noise intensity, with slower deterioration seen for frequencies with more exposure to vocalization noise. Specifically, communication vocalizations were louder at lower frequencies with better hearing thresholds, and these frequencies were less affected by age. We show these results in a new figure 5B and supplementary figure number 1 and we discuss them in the discussion (page 16-17).
The hearing range of this species can be learned from the audiogram presented in Figure 1D. As can be learned from this figure, the frequencies that we used encompasses the main hearing range of adult Rousettus aegyptiacus.
-How the CM have been recorded is unclear to me. An electrode is usually placed next to the tympanic membrane or into the scala tympani. Please provide a detailed description of the procedure.

The reviewer is thinking of an invasive measurement of the CM as is typically done in
animals, but in humans, non-invasive CM measurements are common. We added more information about the procedure in the Methods (page 25) and added the relevant references. In brief: CM were obtained similarly to ABRs, with some modifications: The bats were presented with a 0.1 msec click that was transferred via a 30 cm long air tube to temporally distinguish between physiological CM and speaker artifacts, and the signals were played separately in condensation and rarefaction polarities (because alternating polarity that is used in regular ABR recordings cancels out the CM).

Our method is similar to the one used in humans in which the
-The cochlear histology should be improved: Spiral gg neuron density and the presence of hair cells in the epithelium along the tonotopical axis should be imaged (from histology) and quantified. All results could originate from the degenerescence of hair cells and spiral ganglion neurons.

We thank the reviewer for this comment. We have now added a supplementary image (number 3) showing the spiral ganglion neurons and the hair cells of an aged bat (11.3
years). Both cell types seem intact, however, due to technical difficulties, we could not reliably quantify these cells' survival since they require multiple serial slices. Therefore, we cannot exclude a contribution of possible degeneration of hair cells and spiral ganglion neurons to the hearing phenotype of aged bats at this point.

Minor:
-Different fonts in the sentence : : "In addition, we recorded cochlear microphonics (CM) noninvasively, using the same subdermal electrode positioning used for the ABR recordings…" We fixed the text font.
-In significance statement, space missing between > 10 gr -Check space between value and unit (ex 8kHz shoukd be 8 kHz) We added the space in the significance statement and also checked space between value and unit along the manuscript.
Re is for relative, we added it the first time dB SPL was mentioned in the results.
what is a GLME?
We replaced the term GLME with GLM and added "generalized linear model" the first time we mentioned the abreviation.

Reviewer #2
I suggest changing the title and any use of the term, "suffer," to describe presbycusis in Rousettus. I suggest using "Bats experience.." instead of "Bats suffer from..." throughout the manuscript.
I suggest changing "noninvasive" to "minimally-invasive" for the ABR and CM recordings in We accept and thank the reviewer for these comments, we performed the suggested changes. Thank you for submitting your revised manuscript entitled "Bats experience age-related hearing loss (presbycusis)". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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